BR112019013539A2 - PROPHYLACTIC AND / OR THERAPEUTIC AGENT FOR HEPATIC DISEASE. - Google Patents

Abstract

the present invention addresses the problem of providing a medicinal composition and a drug combination by which non-alcoholic fatty liver disease and non-alcoholic steatohepatitis can be prevented and / or treated. the present invention provides a combination of a peroxisome proliferator receptor activated agonist (ppar) with a sodium and glucose cotransporter 2 inhibitor (sglt2), which should be used to prevent and / or treat non-alcoholic fatty liver disease and steat - non-alcoholic hepatitis.

Description

Contrais Compos’d Tofcgüfozina Compound 1

Toíoglilozina

1/20

PROPHYLACTIC AND / OR THERAPEUTIC AGENT FOR HEPATIC DISEASE

TECHNICAL FIELD [001] The present invention relates to a prophylactic and / or therapeutic agent for non-alcoholic fatty liver disease.

BASICS OF THE TECHNIQUE [002] Non-alcoholic fatty liver disease (NAFLD) is a liver disorder not associated with alcoholic hepatitis or viral hepatitis. NAFLD is estimated to affect about 30% of the general population. NAFLD is a generic term for a range of conditions, from simple steatosis to non-alcoholic steatohepatitis (NASH). Simple steatosis is an accumulation of fat in hepatocytes and has a relatively good prognosis. NASH includes both a fatty liver and inflammation of the liver and can lead to a relatively severe condition such as fibrosis of the liver tissue, liver cirrhosis or hepatocellular carcinoma. Treatment for viral hepatitis such as hepatitis C has made rapid progress recently and therefore the number of patients with liver cancer caused by viruses is expected to decrease in the future. On the contrary, the number of patients with NASH-based liver cancer is expected to increase (Non-Patent Documents 1, 2 and 3).

[003] NAFLD is thought to develop by a widely known “two-hit theory” mechanism in which NAFLD progresses from the fat accumulation stage in hepatocytes to the liver inflammation / fibrosis stage (Non-Patent Document 4). In addition, “multiple parallel strikes theory” has recently been proposed, in which several factors are involved in the progression of NAFLD in parallel (Non-Patent Document 5). In the diagnosis of NASH, key factors are hepatocyte ballooning, Mallory-Denk corpuscle and fibrosis. The Japanese Society of Gastroenterology's NAFLD / NASH Clinicai Practice Guideline defines the pathological diagnostic criteria for NASH as “NAFLD having degeneration

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2/20 ballooning hepatocyte with inflammation in addition to macrovesicular fatty alteration ”.

[004] Matteoni et al. classified patients with NAFLD into four stages based on pathological findings in light of their prognosis, and 3 and 4 Types defined as NASH (Non-Patent Documents 3 and 6). Hepatocyte expansion and balloon degeneration are pathological conclusions that indicate that fat accumulation causes cytoskeleton degeneration. These findings are key diagnostic criteria for NASH.

[005] Treatment for NAFLD is mainly based on care around obesity, diabetes, dyslipidemia and hypertension through lifestyle improvements such as diet therapy and exercise therapy. In addition to lifestyle improvement, drug treatments are performed in clinical practice. Drugs for treatment target insulin resistance, lipid metabolism disorder, hypertension or oxidative stress. Drugs used for insulin resistance include insulin sensitizing drugs such as thiazolidine derivatives (pioglitazone, rosiglitazone, etc.) that are binders for a nuclear PPARy receptor involved in improving insulin sensitivity, or drugs based on biguanide (metformin) , etc.). Drugs used for lipid metabolism disorders include fibrate-based drugs (fenofibrate, bezafibrate, etc.) that are PPARa agonists, statin-based preparations or intestinal cholesterol reabsorption inhibitors (ezetimibe, etc.). Drugs used for hypertension include angiotensin II type 1 receptor antagonists (ARBs) (Non-Patent Documents 1 and 3).

[006] Furthermore, for oxidative stress, drugs used as an antioxidant include vitamin E.

[007] An appropriate regimen for a patient can be selected depending on the underlying disease of these drug treatments. However, any drug treatment requires additional examination.

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3/20

Unfortunately, NAFLD does not currently have any established drug treatment available.

[008] With regard to drugs based on fibrate, fenofibrate is reported to be investigated for the effect with respect to NAFLD in clinical experiments (Non-Patent Document 7). Furthermore, Patent Document 1 describes that (R) -2- [3 - [[N- (benzoxazol-2-yl) -N-3- (4methoxyphenoxy) propyl] aminomethyl] phenoxy] butyric acid or a salt thereof , or a solvate of the same has a selective PPARa activation effect, and Patent Document 2 describes that the compound is useful for preventing and treating non-alcoholic fatty liver disease. However, as for inhibitors of sodium and glucose cotransporter 2 (SGLT2), Remogurifurojin conducted clinical investigation and was reported to have a curative effect on NASH (Non-Patent Document 8). Along with an increase worldwide in patients with metabolic syndrome, the number of patients with NASH is also expected to increase. Since NASH is considered to be a cause of non-viral hepatocellular carcinoma, which is a major factor in cancer-related death (Non-Patent Document 9), it is desired that more effective treatment be established.

Citation List

Patent Document [009] Patent Document 1: WO 2005/023777

Patent Document 2: WO 2015/005365

Non-Patent Document

Non-Patent Document 1: Chalasani N. et al. Hepatology, 55, 2005-23 (2012)

Non-Patent Document 2: Musso G. et al. Nat. Rev. Drug Discov. 15 (4), 249-74 (2016.1)

Non-Patent Document 3: NAFLD / NASH Clinical Practice

Guideline, 2014 of the Japanese Society of Gastroenterology

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Non-Patent Document 4: Day CP. et al, Gastroenterology, 114 (4), 842-5 (1998)

Non-Patent Document 5: Tilg H. et al. Hepatology, 52, 183646 (2010)

Non-Patent Document 6: Matteoni CA. et al. Gastroenterology, 116, 1413-9 (1999)

Non-Patent Document 7: Fernandez-Miranda C. et al. Dig. Liver Dis., 40, 200-5 (2008)

Non-Patent Document 8: Wilkison W. et al. Abstract 0047. International Liver Congress; April 22-26, 2015

Non-Patent Document 9: Fujii M. et al. Med. Mol. Morphol, 46, 141-52 (2013)

Summary of the Invention

Technical Problem [0010] An objective of the present invention is to provide a pharmaceutical composition and / or a combination of drugs that have a preventive and / or ameliorating effect on the expansion of lipid droplets in hepatocytes and / or hepatocyte ballooning and, therefore, that are able to prevent and / or treat NAFLD and NASH.

Solution to the Problem [0011] In view of the aforementioned problems, in order to find a useful means for preventing and / or treating non-alcoholic fatty liver disease (NAFLD), in particular, severe non-alcoholic steatohepatitis (NASH), the present inventors conducted intensive studies using a NASH-HCC mouse model. They observed that a combination of (R) -2- [3 - [[N- (benzoxazol-2-yl) -N-3- (4methoxyphenoxy) propyl] aminomethyl] phenoxy] butyric acid (hereinafter also referred to as Compound 1) which is a PPARa agonist with an SGLT2 inhibitor, which is described in Expert Opin. Investig. Drugs (2013) 22 (4): 463-486 etc.,

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5/20 exerts strong effects to reduce the lipid droplet size in hepatocyte and / or to suppress hepatocyte ballooning and, as a result, prevent and / or treat NAFLD and NASH. The present invention was made on the basis of these findings.

[0012] In other words, the present invention relates to a composition, kit or the like, distinguished by a combination of a PPARa agonist and an SGLT2 inhibitor. More specifically, the present invention relates to the following items [1] to [52].

[0013] [1] A prophylactic and / or therapeutic agent for liver disease, including a combination of a PPARa agonist with an SGLT2 inhibitor.

[0014] [2] The prophylactic and / or therapeutic agent according to item [1], in which the liver disease comprises non-alcoholic fatty liver disease.

[0015] [3] The prophylactic and / or therapeutic agent according to item [2], in which the non-alcoholic fatty liver disease comprises non-alcoholic steatohepatitis.

[0016] [4] The prophylactic and / or therapeutic agent according to item [2], having an effect of suppressing hepatocyte ballooning in a subject with non-alcoholic fatty liver disease.

[0017] [5] The prophylactic and / or therapeutic agent according to item [1], in which the liver disease comprises liver cirrhosis or hepatocellular carcinoma.

[0018] [6] The prophylactic and / or therapeutic agent according to any of the items [1] to [5], in which the PPARa agonist is acid (R) -2 [3 - [[N- (benzoxazole -2-yl) -N-3- (4methoxyphenoxy) propyl] aminomethyl] phenoxy] butyric or a salt thereof, or a solvate thereof.

[0019] [7] The prophylactic and / or therapeutic agent according to

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6/20 any one of items [1] to [6], in which the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, ipragliflozin, empagliflozin, luseogliflozin, tofogliflozina, ertugliflozina, sotagliflozina, bexagliflozina, bexagliflozina, remxagliflozina, bexagliflozina, bexagliflozina, bexagliflozina, remxagliflozina, bexagliflozina, remxagliflozina. [0020] [8] The prophylactic and / or therapeutic agent according to any of the items [1] to [7], being a combination drug.

[0021] [9] The prophylactic and / or therapeutic agent according to any of the items [1] to [7], being a kit.

[0022] [10] A drug for use in the prevention and / or treatment of liver disease, including a combination of a PPARa agonist and an SGLT2 inhibitor.

[0023] [11] The medication according to item [10], in which the liver disease comprises non-alcoholic fatty liver disease.

[0024] [12] The medication according to item [11], in which non-alcoholic fatty liver disease comprises non-alcoholic steatohepatitis.

[0025] [13] The medication according to item [11], having an effect of suppressing hepatocyte ballooning in a subject with non-alcoholic fatty liver disease.

[0026] [14] The medication according to item [10], in which the liver disease comprises liver cirrhosis or hepatocellular carcinoma.

[0027] [15] The medication according to any of the items [10] to [14], in which the PPARa agonist is acid (R) -2- [3 - [[N- (benzoxazole-2il) - N-3- (4-methoxyphenoxy) propyl] aminomethyl] phenoxy] butyric or a salt thereof, or a solvate thereof.

[0028] [16] The medicine according to any of the items [10] to [15], in which the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, ipragliflozin, empagliflozin, luseogliflozina, tofogliflozina, ertugliflozina, sotaglifzzina, sotaglifzzina, sotaglifzina remogliflozin.

[0029] [17] The medication according to any of the items

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7/20 [10] to [16], being a combination drug.

[0030] [18] The medication according to any of the items [10] to [16], being a kit.

[0031] [19] A pharmaceutical composition for preventing and / or treating liver disease, including a PPARa agonist, an SGLT2 inhibitor and a pharmaceutically acceptable carrier.

[0032] [20] The pharmaceutical composition according to item [19], in which the liver disease comprises non-alcoholic fatty liver disease.

[0033] [21] The pharmaceutical composition according to item [20], in which the non-alcoholic fatty liver disease comprises non-alcoholic steatohepatitis.

[0034] [22] The pharmaceutical composition according to item [20], having an effect of suppressing hepatocyte ballooning in a subject with non-alcoholic fatty liver disease.

[0035] [23] The pharmaceutical composition according to item [19], in which the liver disease comprises liver cirrhosis or hepatocellular carcinoma.

[0036] [24] The pharmaceutical composition according to any of items [19] to [23], wherein the PPARa agonist is (R) -2- [3 - [[N (benzoxazol-2-yl ) -N-3- (4-methoxyphenoxy) propyl] aminomethyl] phenoxy] butyric or a salt thereof, or a solvate thereof.

[0037] [25] The pharmaceutical composition according to any of items [19] to [24], in which the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, ipragliflozin, empagliflozin, luseogliflozina, tofogliflozina, ertugliflozina, sotugliflozina, sotugliflozina, beotliflozina, or remogliflozin.

[0038] [26] A method for preventing and / or treating liver disease, including the process of administering to a subject in need of treatment an effective amount of a PPARa agonist and the process of administering

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8/20 to the subject an effective amount of an SGLT2 inhibitor.

[0039] [27] The method for prevention and / or treatment according to item [26], in which the liver disease comprises non-alcoholic fatty liver disease.

[0040] [28] The method for prevention and / or treatment according to item [27], in which the non-alcoholic fatty liver disease comprises non-alcoholic steatohepatitis.

[0041] [29] The method for prevention and / or treatment according to item [27], to suppress ballooning of hepatocytes in the subject with non-alcoholic fatty liver disease.

[0042] [30] The method for prevention and / or treatment according to item [26], in which the liver disease comprises liver cirrhosis or hepatocellular carcinoma.

[0043] [31] The method for prevention and / or treatment according to any of the items [26] to [30], in which the PPARa agonist is acid (R) 2- [3 - [[N- ( benzoxazol-2-yl) -N-3 - (4methoxyphenoxy) propyl] aminomethyl] phenoxy] butyric or a salt thereof, or a solvate thereof.

[0044] [32] The method for prevention and / or treatment according to any of the items [26] to [31], in which the SGLT2 inhibitor is selected from dapagliflozina, canagliflozina, ipragliflozina, empagliflozina, luseogliflozina, tofogliflozina, ertugliflozin, sotagliflozin, bexagliflozin or remogliflozin.

[0045] [33] The method for prevention and / or treatment according to any of the items [26] to [32], in which the PPARa agonist and the SGLT2 inhibitor are administered simultaneously.

[0046] [34] The method for prevention and / or treatment according to any of the items [26] to [32], in which the PPARa agonist and the SGLT2 inhibitor are administered separately at intervals.

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9/20 [0047] [35] Use of a PPARa agonist and an SGLT2 inhibitor to manufacture a prophylactic and / or therapeutic agent for liver disease.

[0048] [36] The use according to item [35], in which the liver disease comprises non-alcoholic fatty liver disease.

[0049] [37] The use according to item [36], in which non-alcoholic fatty liver disease comprises non-alcoholic steatohepatitis.

[0050] [38] The use according to item [36], in which the agent has an effect of suppressing hepatocyte ballooning in a subject with non-alcoholic fatty liver disease.

[0051] [39] The use according to item [35], in which the liver disease comprises liver cirrhosis or hepatocellular carcinoma.

[0052] [40] The use according to any of items [35] to [39], where the PPARa agonist is (R) -2- [3 - [[N- (benzoxazol-2-yl ) -N-3- (4methoxyphenoxy) propyl] aminomethyl] phenoxy] butyric or a salt thereof, or a solvate thereof.

[0053] [41] The use according to any of the items [35] to [40], in which the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, ipragliflozin, empagliflozin, luseogliflozina, tofogliflozina, ertugliflozina, sotaglifzzina, sotaglifzina remogliflozin.

[0054] [42] The use according to any of items [35] to [41], where the agent is a combination drug.

[0055] [43] The use according to any of items [35] to [41], where the agent is a kit.

[0056] [44] A combination of a PPARa agonist with an SGLT2 inhibitor to prevent and / or treat liver disease.

[0057] [45] The combination according to item [44], in which the liver disease comprises non-alcoholic fatty liver disease.

[0058] [46] The combination according to item [45], in which the

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10/20 non-alcoholic fatty liver disease comprises non-alcoholic steatohepatitis.

[0059] [47] The combination according to item [45], having an effect of suppressing hepatocyte ballooning in a subject with non-alcoholic fatty liver disease.

[0060] [48] The combination according to item [44], in which the liver disease comprises liver cirrhosis or hepatocellular carcinoma. [0061] [49] The combination according to any of items [44] to [48], wherein the PPARa agonist is (R) -2- [3 - [[N- (benzoxazol-2-yl ) N-3- (4-methoxyphenoxy) propyl] aminomethyl] phenoxy] butyric or a salt thereof, or a solvate thereof.

[0062] [50] The combination according to any of items [44] to [49], in which the SGLT2 inhibitor is selected from dapagliflozin, canagliflozin, ipragliflozin, empagliflozin, luseogliflozina, tofogliflozina, ertugliflozina, sotaglifzina, beotaglifzina, remogliflozin.

[0063] [51] The combination according to any of items [44] to [50], being a combination drug.

[0064] [52] The combination according to any of items [44] to [50], being a kit.

Advantageous Effects of the Invention [0065] The therapeutic agent, drug, pharmaceutical composition, method of treatment, use or combination of the present invention can suppress lipid droplet expansion in hepatocytes and / or hepatocyte balonization in a patient with non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). In this way, the present invention can provide innovative NAFLD and NASH prevention and / or treatment. In particular, the present invention can provide prevention and / or treatment of highly severe NASH.

BRIEF DESCRIPTION OF THE DRAWINGS

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11/20 [0066] FIG. 1 shows the lipid droplet size (gm ² ) in hepatocytes when Compound 1 (0.1 mg / kg), tofogliflozin (10 mg / kg) or a combination of Compound 1 (0.1 mg / kg) and tofogliflozin (10 mg / kg) of the present invention is administered.

[0067] FIG. 2 shows hepatocyte ballooning when Compound 1 (0.1 mg / kg), tofogliflozin (10 mg / kg) or a combination of Compound 1 (0.1 mg / kg) and tofogliflozin (10 mg / kg) of the present invention is administered.

[0068] FIG. 3 shows hepatocyte ballooning when Compound 1 (0.1 mg / kg), ipragliflozin (3 mg / kg) or a combination of Compound 1 (0.1 mg / kg) and ipragliflozin (3 mg / kg) of the present invention is administered.

Description of Modalities [0069] In the present invention, PPARa agonist means a generic term for compounds that activate a PPARa type receptor. PPARa type receptor is a type of peroxisome proliferator (PPAR) activated receptors that are a kind of nuclear receptor. PPARa type receptor involves fat oxidation. Specifically included are fibrates such as fenofibrate, clofibrate, bezafibrate, clinofibrate, ciprofibrate, etofibrate and gemfibrozil, and WY-14643 (pyrinic acid), GW-7647 (2- (4- (2- (l- (l-cyclohexane butyl) -3-cyclohexylureido) ethyl) phenylthio) -2-methylpropionate), and pemafibrate. (R) -2- [3 - [[N- (benzoxazol-2-yl) -N-3- (4methoxyphenoxy) propyl] aminomethyl] phenoxy] butyric acid (Compound 1), which is also known under the common name pemafibrate , is used in the present invention. It can be produced, for example, according to the method described in WO 2005/023777 or according to any publicly known method described in Documents.

[0070] In the present invention, SGLT2 inhibitor means a generic term for compounds having an inhibitory effect on a cotransporter 2

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12/20 sodium and glucose (SGLT2) involved in glucose reabsorption in the kidney. Specifically included are dapagliflozin, canagliflozin, ipragliflozin (ASP1941), empagliflozin (BI 10773), luseogliflozina (TS-071), tofogliflozina (CSG452), ertugliflozina (PF-04971729), sotagliflozina (EG-4242) (KGT-1681). Each of these compounds can be used as a pharmaceutically acceptable salt and / or solvate as appropriate, but the present invention includes them all.

[0071] Ipragliflozin is a common name for a compound (IS) -1,5 anhydro-1 -C- {3- [(1-benzothiophen-2-yl) methyl] -4-fluorophenyl} -D-glucitol.

Ipragliflozin can be used, for example, as ipragliflozin L-proline (1: 1).

[0072] Tofogliflozina is a common name for a compound (1S, 3'R, 4'S, 5'S, 6'R) -6 - [(4-ethylphenyl) methyl] -6 '- (hydroxymethyl) -3', 4 ' , 5 ', 6'-teetrahydro-3H-spiro [2-benzofuran-1,2'-pyran] -3', 4 ', 5'-triol. Tofogliflozin can be used, for example, as tofogliflozin monohydrate.

[0073] Ipragliflozin can be produced, for example, according to the method described in WO 2004/080990 or according to any publicly known method described in Documents.

[0074] Tofogliflozin can be produced, for example, according to the method described in WO 2006/080421 or according to any publicly known method described in Documents.

[0075] In the present invention, liver disease includes fatty liver, hepatitis, NAFLD, NASH, liver cirrhosis and liver cancer such as hepatocellular carcinoma.

[0076] The "salt" of the present invention is not particularly limited, as long as it is pharmaceutically acceptable. It includes alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; inorganic based salts such as salt

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13/20 ammonium; organic based salts such as trialkylamine salt; mineral acid salts such as hydrochloride salt and sulfate salt; and salts of organic acid such as acetate salt.

[0077] The "solvate" of the present invention includes a hydrate and an alcoholate (such as ethanolate).

[0078] A combination drug is a pharmaceutical product including two or more active ingredients in a single dosage form. In the present invention, an example of the combination drug can be a tablet including the PPARa agonist and the SGLT2 inhibitor in an effective amount.

[0079] A kit is a set of two or more pharmaceutical products. Each of the pharmaceutical products can be taken or administered simultaneously or separately at intervals. In the present invention, an example of the kit can be a combination of a pharmaceutical product containing an effective amount of the PPARα agonist with a pharmaceutical product containing an effective amount of the SGLT2 inhibitor.

[0080] NASH is distinguished by hepatocyte ballooning in addition to expansion of lipid droplets in hepatocytes (see Non-Patent Documents 3 and 6). As described in the Examples below, the combined use of Compound 1 with ipragliflozin significantly inhibited hepatocyte ballooning in a NASH-HCC mouse that is a model animal for NASH. Furthermore, the combined use of Compound 1 with tofogliflozin significantly inhibited hepatocyte ballooning in a NASH-HCC mouse and inhibited lipid droplet expansion in hepatocytes. Accordingly, the combined use of the PPARα agonist with the SGLT2 inhibitor of the present invention is useful as a prophylactic and / or therapeutic agent for NASH in mammals including humans.

[0081] The therapeutic agent, medication and the like obtained by combining the PPARa agonist with the SGLT2 inhibitor of the present

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The invention can be used alone or in combination with other pharmaceutically acceptable carriers to prepare a dosage form such as tablets, capsules, granules, powders, lotions, ointments, injections or suppositories. These preparations can be produced according to a known method.

[0082] Therapeutic agents, medicaments and the like obtained by combining the PPARa agonist with the SGLT2 inhibitor of the present invention are administered orally or parenterally. Experienced in the technique can appropriately set the dose depending on a patient's body weight, age, sex, symptom and the like. When Compound 1 as a PPARa agonist is administered to an adult, the daily dose can vary from 0.01 to 1,000 mg, preferably from 0.01 to 10 mg, more preferably from 0.05 to 5 mg, and it is preferably administered in 1 to 3 divided doses. When ipragliflozin is used as an SGLT2 inhibitor, the daily dose can vary from 0.1 to 1,000 mg, preferably from 1 to 200 mg. When tofogliflozin is used as the SGLT2 inhibitor, the daily dose can vary from 0.1 to 500 mg, preferably from 1 to 100 mg. In any case, the daily dose is administered in 1 to 3 divided doses.

Examples [0083] In the following, the present invention is described more specifically with reference to the Examples, but the present invention is not absolutely limited to the Examples.

Example 1 Effect of Compound 1 combined with Tofogliflozin in the NASH Mouse Model [0084] A NASH-HCC mouse is a model animal that develops fatty liver liver cirrhosis through NASH when it is fed a high fat diet, and develops subsequently hepatocellular carcinoma (Non-Patent Document 9). The effect of compound 1 combined with tofogliflozin was examined using the

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15/20 NASH-HCC mouse model.

[0085] In that examination, Compound 1 was prepared according to the method described in Patent Document 1. Tofogliflozin monohydrate (Chugai Pharmaceutical Co., Ltd.) was used as a concrete example of tofogliflozin.

1) Animal used:

[0086] NASH-HCC mice were prepared with reference to Non-Patent Document 9, and were used for this experiment. Specifically, male C57BL / 6J mice were administered subcutaneously with 200 gg of streptozotocin on the second day after birth. The mice were fed ad libitum on a high fat diet (HFD-32, Clea Japan, Inc.) for 2 weeks starting at 4 weeks of age. The mice were separated into four groups as described in 2) below, and were administered with drugs, from 6 weeks of age to 9 weeks of age as described in 3) below. The daily food consumption of the mice in each group was measured while the drugs were administered in such a way that the mice in all groups were fed the same amount of food as the control group.

2) Way of grouping:

[0087] NASH-HCC mice were separated into four groups that included a control group, a group administered 0.1 mg / kg of Compound 1, a group administered 10 mg / kg tofogliflozin and a group administered the combination ( 0.1 mg / kg of Compound 10 mg / kg of tofogliflozin) (n = 5 to 7). The mice were separated immediately before starting drug administration (at 6 weeks of age). There was no difference in the mean body weight between the groups.

3) Drug administration:

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16/20 [0088] The control group was administered with a 3% aqueous solution of gum arabic. Compound 1 and / or tofogliflozin was dissolved in 3% aqueous gum arabic solution, and the group administered with Compound 1, the group administered with tofogliflozin or the group administered with the combination, respectively. The mice in each group were administered orally with the solution in 5 mL / kg of body weight once a day. The administration period was for 3 weeks starting at 6 weeks of age as previously described.

4) Observation and Examination Method:

After the administration period, the liver of each mouse was excised under anesthesia and was fixed with a neutral 10% buffered formalin solution to prepare specimens stained with hematoxylin-eosin. Using the specimens, the lipid droplet size in hepatocyte was analyzed by Image J. image analysis software. The area of 6,000 to 10,000 lipid droplets was measured in each specimen. The median lipid droplet area was calculated for each mouse and then the median and quartile were calculated for each group based on the median for each mouse in the group. These data were expressed in a box and mustache diagram (FIG. 1).

[0089] Hepatocyte ballooning in each mouse was scored under blind conditions according to the following criteria (Kleiner et al., Hepatology 41, 1313-21,2005).

[0090] No. of balloon cells: 0 [0091] Balloon cell is rare (Few balloon cells): 1 [0092] Many balloon cells or prominent (Ballooning): 2 [0093] So the average value of scores (ballooning scores) were calculated in each group and the values were shown (FIG.

2).

[0094] Statistical processing was performed using software

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17/20 statistical EZR, which extends the functions of R and R comander. The software is distributed free of charge on the Jichi Medical University Saitama Medical Center website (Bone Marrow Transplantation (2013) 48, 452-458). The Steel test (N = 5 to 7) was performed using EZR, the drug group administration was marked with the * mark in the figures when the group had a significant difference at p <0.05 with respect to the control group, and marked with the mark ** when the group had a significant difference at p <0.01.

5) Result [0095] As shown in FIG. 1, the median lipid droplet size was 1.17 (μηι ² ) in the control group, 1.00 (μηι ² ) in the group administered with 0.1 mg / kg of Compound 1 and 1.00 (μηι ² ) in the group administered 10 mg / kg tofogliflozin. Although there was a tendency for the size of lipid droplets in hepatocytes to decrease, there was no statistically significant difference. In contrast, with the single-administration groups, the median lipid droplet size was 0.89 (μηι ² ) in their combination use group, and a significant decrease (p = 0.037) was observed in relation to the control group.

[0096] Furthermore, as shown in FIG. 2, the ballooning score was 1.33 in the control group, 0.60 in the group administered 0.1 mg / kg of Compound 1 and 0.71 in the group administered 10 mg / kg of tofogliflozin. Although there was a tendency for hepatocyte ballooning to be suppressed, there was no statistically significant difference. In contrast, with the single-administration groups, the ballooning score was 0.14 in their combination use group, and a notable (p = 0.0089) suppression of hepatocyte ballooning was observed in relation to the control group.

Example 2 Effect of Compound 1 combined with Ipragliflozin in the Mouse Model with NASH [0097] The effect of Compound 1 combined with ipragliflozin was

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18/20 examined in mice with NASH-HCC (Non-Patent Document 9). [0098] In that examination, Compound 1 was prepared according to the method described in Patent Document 1. Ipragliflozin (Shanghai Haoyuan Chamexpress Co., Ltd., Shanghai, China) was used as a concrete example of ipragliflozin.

1) Animals used:

[0099] Mice with NASH-HCC were prepared in the same way as in 1) of Example 1, and were used for this experiment.

2) Grouping way:

[00100] Mice with NASH-HCC were separated into four groups that included a control group, a group administered with 0.1 mg / kg of Compound 1, a group administered with 3 mg / kg of ipragliflozin and a group administered with the combination (0.1 mg / kg of Compound 1 and 3 mg / kg of ipragliflozin) (n = 8). The mice were separated immediately before starting drug administration (at 6 weeks of age). There was no difference in the mean body weight between the groups.

3) Drug administration:

[00101] The control group was administered with 3% aqueous gum arabic solution. Compound 1 and / or ipragliflozin was dissolved in 3% aqueous gum arabic solution, and the group administered with Compound 1, the group administered with ipragliflozin or the group administered with the combination, respectively. The mice in each group were administered orally with the solution in 5 mL / kg of body weight once a day. The administration period was for 3 weeks starting at 6 weeks of age.

4) Observation and Examination Method:

[00102] Observation and examination were carried out in the same way as in 4) of Example 1.

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19/20

5) Result [00103] As shown in FIG. 3, the ballooning score was 1.50 in the control group, 1.35 in the group administered 0.1 mg / kg of Compound 1 and 0.88 in the group administered with 3 mg / kg of ipragliflozin. Although there was a tendency for hepatocyte ballooning to be supplied, there was no statistically significant difference. In contrast, with the single administration group, the ballooning score was 0.38 in its combined use group, and a noted suppression (p = 0.009) of hepatocyte ballooning was observed in relation to the control group.

[00104] As is evident from the previous results, a combined use of Compound 1 with an SGLT2 inhibitor such as tofogliflozin or ipragliflozin of the present invention provides a remarkable effect to reduce lipid droplet size in hepatocytes and suppress hepatocyte ballooning in the model mouse with NASH. Reduction of the lipid droplet size in hepatocytes and suppression of hepatocyte ballooning corresponds to an improvement in the condition of NAFLD and NASH, which in turn leads to the prevention of liver cirrhosis and hepatocellular carcinoma, which are terminal diseases of NAFLD and NASH.

[00105] As is evident from the previous results, a combined use of Compound 1 with an SGLT2 inhibitor such as tofogliflozin or ipragliflozin of the present invention provides a notable effect to reduce lipid droplet size in hepatocytes and suppress hepatocyte ballooning in the model mouse with NASH. Reduction of the lipid droplet size in hepatocytes and suppression of hepatocyte ballooning corresponds to an improvement in the condition of NAFLD and NASH, which in turn leads to the prevention of liver cirrhosis and hepatocellular carcinoma, which are terminal diseases of NAFLD and NASH.

Industrial Applicability [00106] Since a combination of a PPARa agonist and

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20/20 an SGLT2 inhibitor exhibits a prophylactic and / or therapeutic effect on NAFLD and NASH, the present invention has industrial applicability.

Claims (9)

1. Prophylactic and / or therapeutic agent for liver disease, characterized by the fact that it comprises a combination of a PPARa agonist and an SGLT2 inhibitor. 2. Prophylactic and / or therapeutic agent according to claim 1, characterized by the fact that the liver disease comprises non-alcoholic fatty liver disease. 3. Prophylactic and / or therapeutic agent according to claim 2, characterized by the fact that the non-alcoholic fatty liver disease comprises non-alcoholic steatohepatitis. 4. Prophylactic and / or therapeutic agent according to claim 2, characterized by the fact that it has an effect of suppressing hepatocyte ballooning in a subject with non-alcoholic fatty liver disease. 5. Prophylactic and / or therapeutic agent according to claim 1, characterized by the fact that the liver disease comprises liver cirrhosis or hepatocellular carcinoma. Prophylactic and / or therapeutic agent according to any one of claims 1 to 5, characterized in that the PPARa agonist is (R) -2- [3 - [[N- (benzoxazol-2-yl) acid -N-3- (4methoxyphenoxy) propyl] aminomethyl] phenoxy] butyric or a salt thereof, or a solvate thereof. Prophylactic and / or therapeutic agent according to any one of claims 1 to 6, characterized in that the SGLT2 inhibitor is selected from the group consisting of dapagliflozin, canagliflozin, ipragliflozin, empagliflozin, luseogliflozina, tofogliflozina, ertugotliflozina, , bexagliflozina and remogliflozina. 8. Prophylactic and / or therapeutic agent according to any one of claims 1 to 7, characterized by the fact that it is a drug of Petition 870190060643, of 06/28/2019, p. 30/34 2/2 combination. 9. Prophylactic and / or therapeutic agent according to any one of claims 1 to 7, characterized by the fact that it is a kit.